1. Field of the Invention
This invention is related to methods and systems for gas phase sample preparation and introduction into a mass analysis unit.
2. Description of the Related Art
Tandem mass spectrometry (MS/MS) currently plays a central role in the identification and characterization of proteins. Successful mass spectrometric analysis of peptides and proteins relies on the ability to systematically dissect peptide backbone bonds (MS/MS). Conventional MS/MS methods, using collision-activated dissociation (CAD) where ion fragmentation is activated by collisions with buffer gas, fail in this regard if the peptide is too long (approximately >20) residues or contains either labile post-translational modifications or multiple basic residues. Moreover, while intact proteins can be dissociated with CAD, this process routinely produces only a few backbone cleavages making sequence identification challenging.
A different method (compared to CAD) for peptide ion dissociation referred to as electron capture dissociation (ECD) has been developed. In that work, low energy electrons are reacted with peptide cations in the magnetic field of a Fourier transform ion cyclotron resonance MS (FT-ICR-MS). The reaction results in the attachment of electrons to the protonated peptides producing peptide cations containing an additional electron. The odd electron peptide then undergoes very rapid (i.e., femtoseconds) rearrangement with subsequent dissociation. Unlike the collision-activated process, ECD does not cleave chemical modifications from the peptide, but rather induces random breakage of the peptide backbone cleavage that is indifferent to either peptide sequence or length. ECD fragmentation is not limited by the size of the peptide being analyzed. Up to now, fragmentation by ECD could only be performed in expensive (FT-ICR) mass spectrometer.
A further method of fragmentation, known as electron-transfer dissociation (ETD), has been recently introduced. In this method, ECD-like reactions are obtained using negatively charged ions (anions) as vehicles for electron delivery. Given the appropriate anion, the reaction should proceed to donate an electron to the peptide. Subsequently, the peptide would contain an extra electron, and that inclusion of an extra electron is expected to induce peptide backbone fragmentation, just as in ECD. Gas phase peptide cations and small organic anions react rapidly with easily controlled duration and timing. As in ECD, labile post-translational modifications remain intact, while peptide backbone bonds are cleaved with relatively little concern to peptide sequence, charge, or size. Unlike ECD, electron-transfer dissociation (ETD) can be performed with lower-cost bench-top mass spectrometers on a time scale that permits coupling with online chromatographic separations. ETD, however, has two analytical disadvantages compared to ECD: (1) ETD efficiency for doubly charged precursors is lower than with ECD; (2) ETD, which is less energetic, does not induce secondary fragmentation, thus rending the possibility to distinguish the isomeric Leu and Ile residues.
One alternative method for peptide ion dissociation with fragmentation patterns similar to ECD/ETD techniques has been developed. In this method, the peptide cations and anions are stored in radiofrequency (RF) ion traps and irradiated by a beam of metastable species (Ar or He) generated by glow discharged source Fast Atom Bombardment (FAB) gun. These metastable (neutral) species can donate an electron to the peptide cation inducing peptide backbone cleavage the same way as in ECD. An interaction of metastable species with negative peptide ions results in a transfer of electronic excitation and subsequent detachment of an electron from the anion inducing peptide fragmentation. Similar to ECD and ETD, the metastable-induced dissociation does not cleave chemical modifications from the peptide, but rather induces random breakage of the peptide backbone. The major advantage of metastable-induced dissociation is its simplicity. The neutral metastable species can be easily introduced through RF field to the areas where peptide ions are located. However, this method (at least in the current configurations) also encounters problems related to the fragmentation efficiency that is significantly lower than in the conventional ETD.
Background references to these techniques and others related to ion/ion and ion/molecule reactions at high pressure and atmospheric pressure photoionization are listed below, the entire contents of which are incorporated herein by reference.    1. Kaiser, R. E. et al Rapid Comm. Mass Spectrom. 1990, 4, 30);    2. Baba, T. et al Chem. 2004, 76, 4263-4266;    3. Zubarev, R. A. et al J. Am. Chem. Soc. 1998, 120, 3265-3266;    4 Syka, J. E. P. et al PNAS 2004, 101, 9528-9533;    5. Pitteri, S. J. et al. Anal. Chem. 2005, 77, 5662-5669;    6. Chrisman, P. A. et al J. Am. Soc. Mass Spectrom. 2005, 16, 1020-1030;    7. Zubarev, R. A., Principles of mass spectrometry applied to biomolecules, ed. J. Laskin and C. Lifshitz. 2007: Wiley;    8. Misharin, A. S. et al. Rapid Comm. Mass Spectrom. 2005, 19, 2163-2171;    9. Berkout, V. D., Anal. Chem. 2006, 78(9), 3055-3061;    10. Sparkman O. D. et al, US Pat. Appl. Publ. No. US 2006/0250138 A1;    11. Ogorzalek Loo, R. R. et al J. Am. Soc. Mass Spectrom. 1992, 3, 695-705;    12. Pui, D. Y. H. et al U.S. Pat. No. 5,992,244;    13. Stephenson, et al J. Mass Spectrom. 1998, 33 664-672;    14. Ebeling, D. D. et al Anal. Chem. 2000, 72, 5158-5161;    15. Ebeling, D. D. et al U.S. Pat. No. 6,649,907);    16. Whitehouse, G. et al. U.S. Patent Application Pub. No 2006/0255261;    17. Delobel, A. et al Anal. Chem. 2003, 75, 5961-5968;    18. Debois, D. et al J. Mass Spectrom. 2006, 41, 1554-1560); and    19. Demirev, P. A., Rapid Comm. Mass Spectrom. 2000, 14, 777.